负载RPL8蛋白的DC疫苗抑制乳腺癌生长的实验研究

The dendritic cells loaded with RPL8 protein inhibits the growth of breast cancer in mice

目的制备负载RPL8蛋白的DC疫苗,探讨DC疫苗对小鼠乳腺癌生长抑制作用及作用机制。方法用PCR从质粒p UC57-RPL8中扩增RPL8片段,并将该片段插入p ET28α(+)原核表达载体中,实现重组蛋白的表达及纯化。用SDS-PAGE和Western blot对表达产物进行鉴定。分离培养小鼠骨髓细胞来源细胞。RPL8蛋白冲击DC,荧光显微镜下观察DC负载RPL8蛋白情况。MTT法检测淋巴细胞的杀伤效应;将负载RPL8蛋白的DC疫苗注射入乳腺癌荷瘤小鼠体内,观察肿瘤体积变化及小鼠的生存时间。结果成功构建p ET28α(+)-RPL8原核表达载体。经SDS-PAGE和Western blot鉴定,有目的蛋白表达,树突状细胞内有荧光颗粒。负载RPL8蛋白DC疫苗体外杀伤4T1乳腺癌细胞的细胞毒活性与未负载RPL8蛋白的DC及PBS组比较,有显著性差异(P<0.05)。DC疫苗注入荷瘤小鼠体内后,小鼠生存期、肿瘤体积变化较对照组有显著性差异(P<0.05)。结论负载RPL8蛋白的DC疫苗对小鼠乳腺癌具有免疫治疗作用,为乳腺癌的免疫治疗提供理论依据。

This study aim ed to explore the anti-tumor effect of RPL8-DC on breast cancer in mice. RPL8 gene was amplified from plasmid p UC57-RPL8 with PCR, and cloned into p ET28α（＋） prokaryotic expression vector. The recombinant protein was achieved through IPTG induction in E. coli and identified with SDS-PAGE and Western blot, and then loaded on bone marrow-derived DC. RPL8 and CD11 c, CD80, MHC- Ⅰ, MHC- Ⅱmolecules on the dentritic cells were detected by fluorescence microscope and FACS analysis, respectively. The anti-tumor effect of T cells in vitro was detected by MTT assay. Subcutaneous tumors were induced in Balb/c mice using 4T1 cells. The tumor size and survival time of the mice were observed after RPL8-DC injection. DCs pulsed by RPL8 were visualized under fluorescent microscopy. Data showed that CD11 c, CD80, MHC- Ⅰ, MHC- Ⅱmolecules on DCs were up-regulated after stimulation with LPS; 4T1 cells were inhibited in vitro by T cells stimulated with RPL8-DC（P〈0.05）; the inhibition rate of tumor cells was 70% in RPL8-DC group when effectorto-target ratio was 30∶1, which was higher than those of PBS and DC groups. Inhibition of tumor growth was more significantly in mice after the treatment with RPL8-DC（P〈0.05）. The mice receiving the therapy of RPL8-DC were able to survive much longer than the mice receiving control therapy. The result indicated that the DC pulsed by RPL8 protein can inhibit the growth of breast cancer, providing a theoretical basis for the immune therapy of breast cancer.